Preparation for typing of staphylococci and process therefor



United States Patent 3,094,466 PREPARATION FOR TYPING 0F STAPI-IYLOCOCCIAND PROCESS THEREFOR Benjamin S. Schwartz, Livingston, N.J., assignor toWarher-Lambert Pharmaceutical Company, Morris Plains, NJ a corporationof Delaware No Drawing. Filed July 15, 1959, Ser. No. 827,140 4 Claims.(Ci. 195-1035) This invention relates to a new and novel preparationuseful in the routine typing and identification of Staphylococciorganisms and to a method of producing said preparation.

In recent years, a serious problem has developed in hospitals as aresult of infections caused by antibioticresistant strains ofstaphylococcus, and particularly strains of Staphylococci aureus. Thesestrains are harbored not only among professional personnel and patientsbut may also be found in the physical environment of the hospital, forexample on bedding and furniture, in dust or in the air. The presence ofthese antibiotic-resistant strains represents a reservoir which acts asa constant potential source of infection, which is manifested by thefrequent outbreaks of Staphylococci infections among hospitalizedmaternity patients and infants. It is frequently found that a singlestrain is responsible for the major problem in any one hospital andprompt and accurate identification of this particular strain and itssource would greatly aid in controlling this problem.

Bacteriophages, usually known as phages, are viral agents which producetransmissible dissolution of specific bacterial cells. Phage typing ofStaphylococci is a wellknown bacteriological technique used to identifyan unknown strain of this organism. Most strains of an organism, forexample the strains of Staphylococci, are sensitive to a particularphage or group of phages with the result that a culture of an unknownstrain may be typed by observing the action upon the strain of a seriesof known bacteriophages. Where lysis of the unknown strain occurs in thepresence of a known phage or group of phages, it is established that thestrain is sensitive to the particular phage or group of phages andenables one to thereby identify the strain.

Fisk, Roy T., J. Infect. Dis. 71, 161-165 (1942), Williams et al., J. ofHygiene 50, 320-353 1952), and Blair et al., J. Infect. Dis. 93, 1-13(1953) discuss the general principles involved in phage typing ofstrains of Staphylococci. The procedures described, while adapted to useby specialized typing laboratories, are too cumbersome and timeconsuming for routine use in a hospital.

Recently there have been made commercially available lyophilized stablephages of 29 strains of Staphylococci, including the strains mostcommonly encountered in recent hospital infections. These phages are inthe form of dry powders and thus represent a source of stock phageswhich avoid the need for extensive propagation by the laboratory.However, even these preparations present problems in use. Thelyophilized strain must be reconstituted in broth, cultured in thepresence of its propagating strain, harvested and titrated tostandardize the phage content of the filtrate. These procedures must allbe carried out before typing can begin. Test dilutions of the stockfiltrate are not stable for more than a few days and frequentrestandardization is required. Thus, even these preparations do notsolve the problem of providing a tool for rapid typing which may becarried out within a hospital laboratory without highly specializedtraining.

It is apparent that effective control of an outbreak of staphylococcalinfection depends upon prompt identification of the strain responsibleand also the location of carriers or other sources of the organismwithin the hospital community.

It is, therefore, a particular object of this invention to provide apreparation adapted to the rapid typing of Staphylococci and which maybe used on a routine basis without highly specialized training.

Other objects and the advantages of this invention will become apparentfrom the following detailed description.

In accordance with this invention semi-porous solid beads areimpregnated with high titer filtrate of a specific staphylococcal phageand the impregnated beads are then dried to provide beads eachcontaining a specific quantity of viable phage organisms. In use, one ormore of these impregnated beads are shaken with an appropriate liquidmedium, thereby providing a standardized solution containing thespecific phage. This phage solution may then be used directly in typingby applying a drop to a culture of the unknown organism and observingWhether lysis of the organism occurs.

The initial step in the preparation of the impregnated beads of thisinvention is the preparation of a high titer broth culture filtrate(titer of at least 10 containing a specific staphylococcal phage. Thisstep is conventional and any of the techniques described in the priorart may be used, for example those described by Williams et al., J. ofHygiene 50, 320-353 (1952), Blair et al., J. Infect. Dis. 93, 1-13(1953), and Swanstrom et al., Proc. Soc. Exp. Biol. & Med. 78, 372-375(1951). The phage is recultured repeatedly on its propagating strainuntil serial titrations indicate that a satisfactory potency has beenobtained. The phage suspensions are ordinarily centrifuged at high speedand the supernatant filtered under sterile conditions. Such fresh hightiter filtrates are used to coat the beads.

The typing preparation of this invention is prepared by impregnatingbeads with the high titer filtrate containing a specific staphylococcalphage. The beads used are formed of a semi-porous inert solid material.The term semi-porous as used herein refers to the surfacecharacteristics of the solid material used to form the beads and meansthat the solid material should have the ability to absorb and hold phageorganisms during the impregnation step. Solids having microscopicallyrough surfaces, thus presenting myriad crevices which can hold theorganisms, are desirable. Beads which present a smooth glazed surfaceare generally undesirable since an insufficient number of phageorganisms can be deposited during the impregnation step to provide auseful typing preparation when reconstituted. The term inert as usedherein means that the solid material must contain no substance which iscapable of destroying or impairing the potency of the phage.

A number of solid materials which are inert and which may be formed intosemi-porous beads are available and include synthetic resins, such asphenolic resins, melamine resins and the like, hard rubber, cementitiousor ceramic materials such as unglazed porcelain, and the like. Unglazedporcelain is generally preferred.

The beads may be of any convenient geometric shape, such as spherical,cylindrical and the like. It is desirable that the beads have a maximumexposed surface area for their size. Thus, beads in the shape ofdoughnuts, cups, hollow cylinders, saddles and the like are preferred.With this type of construction, the filtrate containing the phage cancontact both external and internal surfaces during impregnation. Thisinsures that a maximum number of phage organisms are deposited on thebeads.

In typing, small volumes of solutions are normally used. Since theviable phage organisms must be removed from the beads to form a usefultyping solution, it is desirable that the beads be small in size so thatall surfaces thereof may be contacted with the small volume of liquidused in reconstitution. Normally, the maximum dimension of the beadsshould not exceed millimeters.

The titer of the phage filtrate must be carefully controlled to insure aconstant, standard concentration of phage organisms in the impregnatingmedium. In addition, it is apparent that the physical characteristics ofthe beads, such as shape, dimensions, roughness and absorbency should beconstant to insure that the surface of each bead 'is impregnated with auniform number of phage organisms.

The impregnation may *be efiected conveniently by contacting sterilebeads with the filtrate containing the desired phage. The beads shouldpreferably remain in contact with the filtrate for a suflicient time toinsure uniform impregnation of the beads. The impregnated beads are thenremoved from the filtrate.

The final step involves drying the impregnated beads and packaging insuitable containers. Drying may be conveniently effected at roomtemperature under high vacuum or by conventional lyophilization(freeze-drying) techniques. In lyophilization, the wet impregnated beadsare quick frozen and then subjected to an absolute pressure below thevapor pressure of water at the temperature of the frozen beads. Toprevent undue loss in potency during storage, it is essential that theimpregnated beads be completely dry.

The dried impregnated beads may be packaged in several ways, eachadapted to insure complete absence of moisture during storage. Aquantity of beads may be packaged in a sterile vial or tube, with thebeads separated by a porous plug from a mass of a desiccant such assilica gel at the bottom of the containers. Alternately, the sterilevial or tube containing the beads may be packaged within a larger sealedcontainer containing the desiccant. Another convenient packagingtechnique is to seal one or more beads within a length of sterile glasstubing to form a sealed ampule-like container. In this type of package,no desiccant is needed since the sealed glass prevents the entry of anymoisture. In this method of packaging, each ampule-like containercontain the number of beads (one or more) required for a singlereconstitution.

In use, one or more beads are removed aseptically from the container andthoroughly contacted with a liquid. Useful liquids which may be used inreconstitution include water or any general purpose medium of the typeused in culturing staphylococcal phage organisms. A typical generalpurpose medium useful in reconstitution is a broth containing dextrose,salt, buffers and one or more peptones, that is enzymatic hydrolysatesof proteinaceous materials such as soybean meal, casein and the like.During reconstitution, the viable phage organisms with which the surfaceof the beads are impregnated become suspended in the liquid, and theresulting solution may be utilized in typing without the necessity ofculturing and standardizing the phage.

The method of typing after reconstitution of the phage is conventional,the procedure described by Blair et al., J. Infect. Dis. 93, 1-13 (1953)being typical. One agar plate is used for each strain to be typed. Witha swab or loop, the surface of the plate is seeded uniformly from a 4-6hour broth culture of the unknown strain and the inoculation is allowedto dry. With a 4 mm. loop, or a capillary pipette, one loopful or onedrop of reconstituted phage is placed on the seeded area at spacedintervals about 1 cm. apart. After the phages have dried, the plate isinverted and incubated for 18-24 hours at 30 C. From 30-36 phages can bespotted on a single plate. The reactions are recorded according to thedegree of lysis. Strains are differentiated on the basis of theirsensitivity to one or more members of several distinct groups of phages.

The specific advantage of the impregnated beads of the present inventionis that the laboratory wishing to type an unknown strain ofStaphylococci need not carry out the cumbersome and laborious proceduresof culturing and standardizing numerous phages, since the impregnatedbeads may be easily reconstituted and the resulting solution useddirectly in typing.

In some circumstances where it is desired to determine whether an unkownorganism is one of a group of strains of Staphylococci, a pooledreconstituted phage solution may be used. In this technique, a series ofbeads, each impregnated with a specific phage, may be reconstiutedtogether to form a single pooled solution which may then be used intyping. Sufficient phages are represented to insure positiveidentification of the unknown organism as one of the group of strains.

The following examples are included in order further to illustrate thisinvention:

Example 1 Small cup-shaped unglazed porcelain beads (Fish Spine beadsNo. 2 from Taylor, Tunnicliffe and Co., London, England) having amaximum dimension of 4 mm. are immersed in a high titer filtrate ofstaphylococcal phage strain No. 80. The beads are separated asepticallyfrom the filtrate, dried under high vacuum at room temperature, andfinally inserted into sterile tubes containing a mass of silica gelcovered by a sterile glass wool plug. The tubes are then closed withsterile stoppers.

Example II Unglazed porcelain beads are impregated as described inExample I and then subject to lyophilization in a Dry Ice bath. Thelyophilized beads are then packaged as described in Example 1.

Example III Lyophilized beads prepared as described in Example II areplaced within a length of sterile glass tubing having an inside diameterslightly in excess of the size of the beads. The beads are arrangedwithin the tubing in a series of spaced pairs of beads. The glass in thearea of the spaces is sealed to provide a chain of connected sealedampule-like containers, each containing two of the lyophilized beads. Inuse in typing, an ampule may be broken from the chain and the two beadsreconstituted with broth to provide a standardized solution ofstaphylococcal hage, strain No. 80.

It is understood that the foregoing detailed description is given merelyby way of illustration and that many variations may be made thereinwithout departing from the spirit of my invention.

Having described my invention, what I desire to secure by Letters Patentis:

l. A preparation useful in the rapid routine typing of Staphylococciconsisting of an unglazed dried porcelain sad the surface of which isuniformly coated and impregnated with a controlled and calibratedquantity of viable and removable bacteriophage organisms effectiveagainst a strain of Staphylococci.

2. A method of producing a bacteriophage preparation useful in the rapidroutine typing of Staphylococci which comprises immersing a plurality ofbeads of a semi-porous inert solid material into a liquid mediumcomprising a filtrate containing viable bacteriophage organismseffective against a strain of Staphylococci, separating wetted beadsfrom the liquid medium and drying the beads.

3. A method of preparing a solution useful in the rapid routine typingof Staphylococci which comprises contactmg with a liquid at least onedried bead of a semi-porous inert solid material the surface of which isimpregnated with viable bacteriophage organisms effective against astrain of Staphylococci, whereby said viable bacteriophage.

organisms are released from said bead and distributed throughout saidliquid to form said solution.

4. A method of preparing a pooled bacteriophage solution useful in theidentification of an unknown organism as a member of a group of strainsof Staphylococci which comprises contacting with a liquid a plurality ofdried beads of a semi-porous inert solid, the surfaces of said ibeadsbeing impregnated 'with viable bacteriophage organisms effective againstsaid strains of Staphylococci, no single bead being impregnated withmore than a single bacteriophage strain, there being at least oneimpregnated bead for each strain of bacteriophage and sufiicientbacteriophage strains being represented to insure positive typing ofsaid unknown organism as a member of said group, whereby said viablebacteriophage organisms are released from said beads and distributedthroughout said liquid to form said pooled bacteriophage solution.

References Cited in the file of this patent FOREIGN PATENTS GreatBritain Apr. 3, 1957 OTHER REFERENCES Science News Letter, 68:1, July 2,1955, p. 14.

Adams, I. Baot., 72:4, October 1956, p. 572.

Calmon: Ion Exchangers in Organic and Biochemistry, Interscience Pub,New York, 1957, pp. 25 0251.

Fedn. Procs., 17:1, March 1958, No. 252, p.. 64.

Science, 127:3303, April 18, 1958, pp. 859-863.

Barboriak: P.S.E.B.M., 98:2, June 1958, pp. 288-290.

1. A PREPARATION USEFUL IN THE RAPID ROUTINE TYPING OF STAPHYLOCOCCICONSISTING OF AN UNGLZED DRIED PORCELAIN BEAD THE SURFACE OF WHICH ISUNIFORMLY COATED AND IMPREGNATED WITH A CONTROLLED AND CALIBRATEDQUANTITY OF VIABLE AND REMOVABLE BACTERIOPHAGE ORGANISMS EFFECTIVEAGAINST A STRAIN OF STAPHYLOCOCCI.